A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment

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dc.creatorBrown, Marina E.
dc.creatorMontgomery, Micaela L.
dc.creatorKamath, Manali M.
dc.creatorNicholas, Sarah
dc.creatorLiu, Yutao
dc.creatorKaramichos, Dimitrios
dc.creatorFuller, Kevin K.
dc.creator.orcid0000-0002-8761-3824 (Karamichos, Dimitrios)
dc.date.accessioned2022-09-20T20:19:47Z
dc.date.available2022-09-20T20:19:47Z
dc.date.issued2021-04-15
dc.description.abstractFungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models involving co-culture of the pathogen and the corneal cells in tissue culture medium are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFalpha, IL-1beta, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK.
dc.description.sponsorshipThis work was supported by a Research to Prevent Blindness (RPB) Career Development Award to KKF, a RPB Unrestricted Grant to Dean McGee Eye Institute, and a 5P30EY021725- 10 from the National Eye Institute to the OUHSC Department of Ophthalmology.
dc.identifier.citationBrown, M. E., Montgomery, M. L., Kamath, M. M., Nicholas, S., Liu, Y., Karamichos, D., & Fuller, K. K. (2021). A novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment. Experimental eye research, 207, 108581. https://doi.org/10.1016/j.exer.2021.108581
dc.identifier.issn1096-0007
dc.identifier.urihttps://hdl.handle.net/20.500.12503/31792
dc.identifier.volume207
dc.publisherElsevier Ltd.
dc.relation.urihttps://doi.org/10.1016/j.exer.2021.108581
dc.rights.holder© 2021 The Authors.
dc.rights.licenseAttribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.sourceExperimental Eye Research
dc.subjectCorneal fibroblasts
dc.subjectCorneal inflammation
dc.subjectFungal keratitis
dc.subjectFusarium
dc.subjectKeratocytes
dc.subjectMatrix metalloprotease
dc.subject.meshAnimals
dc.subject.meshAspergillosis / metabolism
dc.subject.meshAspergillosis / microbiology
dc.subject.meshAspergillosis / pathology
dc.subject.meshAspergillus fumigatus / physiology
dc.subject.meshCell Culture Techniques
dc.subject.meshCorneal Keratocytes / metabolism
dc.subject.meshCorneal Keratocytes / microbiology
dc.subject.meshCorneal Stroma / metabolism
dc.subject.meshCorneal Stroma / microbiology
dc.subject.meshCorneal Stroma / ultrastructure
dc.subject.meshCorneal Ulcer / metabolism
dc.subject.meshCorneal Ulcer / microbiology
dc.subject.meshCorneal Ulcer / pathology
dc.subject.meshCytokines / metabolism
dc.subject.meshDisease Models, Animal
dc.subject.meshEye Infections, Fungal / metabolism
dc.subject.meshEye Infections, Fungal / microbiology
dc.subject.meshEye Infections, Fungal / pathology
dc.subject.meshFusariosis / metabolism
dc.subject.meshFusariosis / microbiology
dc.subject.meshFusariosis / pathology
dc.subject.meshFusarium / physiology
dc.subject.meshHost-Pathogen Interactions / physiology
dc.subject.meshHumans
dc.subject.meshMale
dc.subject.meshMatrix Metalloproteinases / metabolism
dc.subject.meshMice
dc.subject.meshMice, Inbred C57BL
dc.subject.meshMicroscopy, Electron, Transmission
dc.subject.meshReal-Time Polymerase Chain Reaction
dc.titleA novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment
dc.typeArticle
dc.type.materialtext

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